Screening Of Antioxidative And Probiotic Activities Of Lactic Acid Bacteria And Its Protecting Effect To Oxidative Damage Of Pig Intestinal Epithelial Cell

Weaned piglets diarrhea is an important disease with high disease incidence rate,which affects the development of the pig industry,resulted in serious economic loss,and become a problem in the swine industry worldwide.Enterotoxigenic Escherichia coli?ETEC?is an important factor in causing diarrhea in weaned piglets.The epidemiological survey shows that F4⁺ETEC occupy a dominant position in the diarrhea.Recent studies showed that there showed obvious oxidative stress in the intestinal tract of piglets when weaned piglets diarrhea happened.It has been reported that lactic acid bacteria could relieve oxidative stress,but the mechanism of action remains unclear.From the view of antioxidative process,it is hopeful to discover a new way to improve the effect of prevention and control of weaned piglets diarrhea.The aim of this study was to apply the oxidative stress cell model and the ETEC infected cell model to evaluate the protective effects of the selected probiotic lactic acid bacteria,and to further elucidate the mediating role of Nrf2 signaling pathway in oxidative stress and intestinal protection against ETEC from the cellular and molecular level.The main results are as follows:First,322 strains from traditional artisanal milk cheese from northeast China were isolated,screened and identified,by gram staining,microscopy and catalase test.By phenotypic traits,molecular identification,tolerance analog gastrointestinal tract environment,adsorption ability and probiotic characteristics as evaluation index,a total of 23 strains was preliminary screened.The 23strains had good inhibitory effect on five common pathogens and had strong scavenging ability of free radicals.Subsequently,the 23 strains with excellent probiotics and strong antioxidant capacity were identified by 16S rRNA.The 23 isolates belonged to the species Lactobacillus plantarum?16?,Lactobacillus paracasei?2?,Enterococcus faecium?2?,Lactobacillus helveticus?1?,Weissella paramesenteroides?1?,and Pediococcus pentosaceus?1?.Furthermore,6 strains were screened by safety test,hydrophobicity and antibiotic sensibility.Following the antioxidant activities of the exo polysaccharides,surfactants and small molecular proteins of 6 strains was measured by scavenging ability of free radicals.Among them,L.plantarum 27197 was selected for subsequent experiments with the best probiotic properties and antioxidant capacity.After that,the oxidative stress cell model based on H₂O₂ was established to evaluate the effect of L.plantarum 27197 on oxidative stress and to analyze the mechanism of alleviating oxidative stress.The control group,H₂O₂ group,cell-free culture supernatant of lactic acid bacteria LAB-cell group,cell-free culture supernatant of LAB-H₂O₂-cell group,LAB-cell group,and LAB-H₂O₂-cell group were set up to observe the morphological changes,and detecting oxidative stress and the level of mRNA or proteins expression related to Nrf2 signaling pathway,inflammatory factors and intestinal mucosal barrier.The results showed that L.plantarum 27197could obviously alleviate the damage of oxidative stress,reducing the damage of cell morphology and relieve the colonic inflammation reaction caused by oxidative stress.Secondly,the infecting cells model of ETEC K88 was established,and the mechanism of remission effect of L.plantarum 27197 on intestinal damage was evaluated and analyzed.The control group,K88infection group,cell-free culture supernatant of LAB-cell group,cell-free culture supernatant of LAB-K88-cell group,LAB group and LAB-K88-cell group were established.The mechanism of L.plantarum 27197 alleviated K88 damage.The results showed that K88 has obvious oxidative damage early in intestinal epithelial cells.L.plantarum 27197 can alleviat the effect for the oxidative stress damage caused by K88,and making the related detection index to normal cell group.In addition,L.plantarum 27197 can reduce the transcriptional level of inflammatory factors and improve the transcriptional level of the intestinal tight connexion factor,indicating that L.plantarum 27197 inhibits the inflammatory response induced by K88 in the body and maintains the intestinal mucous membrane barrier.It was also found that L.plantarum 27197 could regulate Nrf2signaling pathway related factors to protect cells and regulate transcription level of K88 virulence factors to reduce ETEC damage to cells.This study has been verified that L.plantarum 27197 can regulate the expression of related antioxidant proteins in the Nrf2 signaling pathway,which can alleviate oxidative stress.The protective mechanism is related to the maintenance of intestinal mucosal barrier function,the regulation of cytokine expression,and the expression of K88 virulence factors.L.plantarum27197 can be used as a safe antioxidant to prevent the oxidative stress caused by K88 in weanling pigsearly diarrhea,which lays a strong foundation for the next step of adding probiotic lactic acid bacteria to animal feed as antioxidants.The experimental results provide a theoretical basis for revealing the mechanism of probiotic lactic acid bacteria with strong antioxidant ability in alleviating the oxidative stress reaction of intestinal epithelial cells and the damage of F4⁺ETEC in the intestinal epithelial cells of weanling piglets.It also provides a theoretical basis for the scientific application of probiotic lactic acid bacteria.